EP0595345B1 - Process for producing alanylglutamine - Google Patents

Process for producing alanylglutamine Download PDF

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Publication number
EP0595345B1
EP0595345B1 EP19930117578 EP93117578A EP0595345B1 EP 0595345 B1 EP0595345 B1 EP 0595345B1 EP 19930117578 EP19930117578 EP 19930117578 EP 93117578 A EP93117578 A EP 93117578A EP 0595345 B1 EP0595345 B1 EP 0595345B1
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glutamine
added
mixture
room temperature
solution
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German (de)
French (fr)
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EP0595345A1 (en
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Kazumi Amatsu
Kunimi Inoue
Masaji Kasai
Yukiteru Mimura
Shoichi Mizutaki
Yasunori Nakaguchi
Yasuyuki Ono
Yutaka Osawa
Hiroyuki Shinmura
Shinji Tomioka
Yoshiyuki Yamada
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Kyowa Hakko Kogyo Co Ltd
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Kyowa Hakko Kogyo Co Ltd
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/63Esters of sulfonic acids
    • C07C309/64Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to acyclic carbon atoms
    • C07C309/65Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to acyclic carbon atoms of a saturated carbon skeleton
    • C07C309/66Methanesulfonates
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/22Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/63Esters of sulfonic acids
    • C07C309/72Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C309/73Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton to carbon atoms of non-condensed six-membered aromatic rings
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06Dipeptides
    • C07K5/06008Dipeptides with the first amino acid being neutral
    • C07K5/06017Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala

Description

    Background of the Invention
  • The present invention relates to a process or producing alanylglutamine and a novel N-(2-substituted)propionylglutamine compound which is an intermediate for the alanylglutamine.
  • L-alanyl-L-glutamine has higher stability and higher water-solubility than L-glutamine, and is used as the component of the infusion solution.
  • For the production of alanylglutamine, there have heretofore been known three types of methods (1), (2) and (3): (1) methods of using a protecting group, for example, the method which comprising condensing an N-benzyloxycarbonylalanine (hereinafter referred to as a "Z-alanine") with a protected glutamine in the presence of dicyclohexylcarbodiimide (DCC), and removing the protecting group from the intermediate compound [Bull. Chem. Soc. Jpn., 34, 739 (1961); Bull. Chem. Soc. Jpn., 35, 1966 (1962)]; the method which comprising condensing a Z-alanine with a protected γ-methyl glutamate in the presence of DCC, removing the protecting group from the intermediate compound and further reacting the deprotected product with ammonia [Bull. Chem. Soc. Jpn., 37, 200 (1964)]; and the method which reacting an active ester of a Z-alanine with a non-protected glutamine and removing the protecting group from the intermediate compound (European Patent No. 311,057); (2) the method for producing alanylglutamine via an N-carboxyl anhydride (German Patent No. 3,206,784); and (3) the method using 2-bromopropionyl chloride as a starting compound via an intermediate compound, 2-bromopropionylglutamine (Hoppe-Seyler's Z. Physiol. Chem., 105, 58 (1919)).
  • The methods (1) using a protecting group need the step of removing the protecting group from the intermediate compound and the operation for the step is complicated. Therefore, the methods (1) yield alanylglutamine at higher cost. The method (2) uses an N-carboxyl anhydride of alanine without involving a protecting group. However, by-products such as tripeptides are considerably produced and the yield of the intended product is lower. In addition, it is difficult to purify the intended product. In the method (3), since an acid chloride having a high reactivity with water is added to an aqueous solution of glutamine for the reaction of 2-bromopropionyl chloride with glutamine, the method involves hydrolysis of the acid chloride. Therefore, the method (3) yields by-products and the yield of the intended product is low. Since the produced 2-bromopropionylglutamine is purified by extraction with an organic solvent, the yield and optical purity of the product are low. In addition, in the method (3), since the ammonolysis of 2-bromopropionylglutamine is carried out at a higher temperature, by-products are considerably produced and the optical purity of the produced alanylglutamine is often low.
  • Summary of the Invention
  • An object of the present invention is to provide a process for producing alanylglutamine and a novel N-(2-substituted)propionylglutamine compound.
  • In accordance with the present invention, there is provided a process for producing alanylglutamine which comprising reacting an N-(2-substituted)propionylglutamine compound represented by the formula (I) [hereinafter referred to as Compound (I); compounds having other formulae numbers are similarly referred to]:
    Figure 00030001
    where X represents halogen, alkylsulfonyloxy, or substituted or unsubstituted arylsulfonyloxy, with ammonia at a temperature of 60°C or below.
  • Further, in accordance with the present invention, there is provided a novel N-(2-substituted)propionylglutamine compound represented by the formula (I'):
    Figure 00040001
    where X1 represents chlorine, or a salt thereof.
  • Detailed Description of the Invention
  • In the definition of the groups in formulae (I) and (I'), the alkyl moiety of the arylsulfonyloxy includes, or example, a straight or branched alkyl having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl; the aryl moiety in the arylsulfonyloxy includes, for example, phenyl and naphthyl; and the substituted aryl includes, for example, tolyl. The halogen includes, for example, chlorine, bromine and iodine.
  • In order to obtain Compound (I), a process for producing Compound (I) which comprises reacting a 2-substituted-propionyl halide represented by the formula (II):
    Figure 00050001
    where X has the same meaning as mentioned above and Hal represents halogen, with a glutamine-containing aqueous alkaline solution in the presence of a water-immiscible organic solvent, can be used. The halogen has the same meaning as mentioned above.
  • The water-immiscible organic solvent to be used in the process for producing an N-(2-substituted)propionylglutamine, includes, for example, ether, toluene, chloroform, methylene chloride, dichloroethane and ethyl acetate. The solvent may be used singly or as a mixture of them. Particularly, toluene, chloroform and methylene chloride are preferably used. The water-immiscible organic solvent is generally used in an amount of from 0.1 to 5 times-volumes, preferably from 0.3 to one time-volume, based on the amount of the glutamine-containing aqueous alkaline solution. The aqueous alkaline solution is not specifically restricted, so long as it does not interfere with the reaction. For instance, an aqueous solution of an inorganic alkaline substance such as sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate or potassium carbonate and an aqueous solution of an organic alkaline substance such as trimethylamine, triethylamine or pyridine are mentioned. Preferably, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate and triethylamine are used. Glutamine to be reacted is used in an amount of from 0.5 to 2.0 equivalents based on the 2-substituted-propionyl halide. One equivalent of glutamine is preferably used. Glutamine to be contained in the aqueous alkaline solution is used in an amount of from 0.01 to 3.0M, preferably from 0.1 to 1M. The reaction is carried out at a temperature of from -5 to 40°C, preferably from 0 to 10°C, in a period of from 0.1 to 5 hours, preferably from 0.5 to 2 hours. During the reaction, the pH value of the aqueous alkaline solution is kept from 7 to 11, preferably from 9 to 10.5. With the progress of the reaction, since hydrochloric acid is formed in the reaction mixture, the pH of the solution is lowered. Therefore, it is preferred to add a base to the reaction mixture during the reaction so as to adjust the pH of the reaction mixture to be within the above-mentioned range. The base to be used for the purpose is not specifically restricted, so long as it does not interfere with the reaction. For instance, inorganic bases such as sodium hydroxide and organic bases such as triethylamine are mentioned. Thus, Compound (I) is formed in a high yield in the aqueous alkaline solution.
  • The water-immiscible organic solvent is removed by liquid separation or the like. Then a salt is added to the resulting aqueous alkaline solution, and the pH of the solution is adjusted to be from 0.1 to 4, preferably from 0.5 to 2.5, with a strong acid such as hydrochloric acid or sulfuric acid, for salting-out of Compound (I) in a high yield. The salt to be used for the purpose includes, for example, sodium chloride, potassium chloride and sodium sulfate. Sodium chloride is preferably used. The amount of the salt to be added is not specifically restricted, and it is preferred that the aqueous alkaline solution may be a saturated solution of the salt.
  • Among the N-(2-substituted)propionylglutamines to be obtained by the above-mentioned method, that represented by the formula (I');
    Figure 00080001
    where X1 represents chlorine,and a salt thereof are novel compounds. The salt of Compound (I') includes, for example, an alkali metal salt such as sodium or potassium salts, an ammonium salt such as ammonium, trimethylammonium or triethylammonium salts, and pyridinium salt.
  • Alanylglutamine is produced in a high yield, by reacting Compound (I) or its salt with ammonia.
    Figure 00080002
  • The reaction is usually carried out in a solvent. As the solvent to be used in the reaction, mention may be made of alcohols such as methanol, ethanol and propanol, as well as water and an aqueous solution of an alkaline substance such as sodium hydroxide or potassium hydroxide. Water is preferably used. Addition of an ammonium salt such as ammonium acetate, ammonium chloride, ammonium sulfate, ammonium bromide or ammonium carbonate to the reaction solution often elevates the reactivity. The reaction is generally carried out in the range of from 0 to 60°C under atmospheric or compressed pressure. The reaction is completed in a period of from 1 to 100 hours, preferably from 4 to 50 hours. The ammonia may be used from 1 to 200 equivalents, preferably from 10 to 50 equivalents, based on Compound (I). The concentration of Compound (I) is from 0.01 to 2M, preferably from 0.1 to 0.6M. The progress of the reaction may be traced by high performance liquid chromatography (HPLC). After the completion of the reaction, the excess ammonia and water are removed from the reaction mixture, for example, by concentration under reduced pressure, and an alcohol, preferably methanol, ethanol or 2-propanol is added to the mixture to afford a highly purified alanylglutamine in a high yield. Where X is bromine in Compound (I), a highly purified alanylglutamine can be produced in a high yield, without racemization by carrying out the reaction at a temperature of 20 to 30°C.
  • Where an optical-active alanylglutamine is desired, an optical-active Compound (II) and an optical-active glutamine may be used. As the case may be, an optical-inactive Compound (II) and an optical-active glutamine may be used to obtain diastereomeric mixtures of Compound (I) or an alanylglutamine, which mixture may be separated and purified by an ordinary method.
  • The present invention will be explained in more detail by way of the following examples, which, however are not intended to restrict the scope of the present invention.
  • Example 1 Production of N-(2-D-chloro)propionyl-L-glutamine:
  • 48.2g (0.33 mol) of L-glutamine was added to 300 mℓ of water and 150 mℓ of toluene at room temperature and cooled to 0 to 5°C. 66 mℓ (0.33 mol) of 5N sodium hydroxide was added thereto to dissolve the L-glutamine therein. To the solution were added 90 mℓ of toluene containing 42.0g (0.33 mol) of 2-D-chloropropionyl chloride having an optical purity of 92.8% ee and 74 mℓ of 5N sodium hydroxide, at 0 to 5°C over a period of 2 hours, with maintaining the pH of the reaction solution to be 10. The mixture was stirred for one hour at 0 to 5°C, and toluene was removed by liquid separation. 60g of sodium chloride was added to the aqueous layer at room temperature. To the solution was added 22 mℓ of concentrated hydrochloric acid at room temperature, and the pH of the mixture was adjusted to be 2.5. Then, crystal seeds were added to the mixture, and the mixture was stirred for 30 minutes. Further, 8 mℓ of concentrated hydrochloric acid was added thereto so that the pH was adjusted to be 1.0. The resulting solution was allowed to stand for one hour at room temperature. The crystals formed were taken out by filtration and dried under reduced pressure to obtain 71.6g (yield: 85.3%, purity: 92.9%) of N-(2-D-chloro)propionyl-L-glutamine having an optical purity of 99.4% de and a melting point (with decomposition) of 148°C.
  • The physico-chemical properties of N-(2-D-chloro)propionyl-L-glutamine were as follows:
       1H-NMR (300 MHz, DMSO-d6) δ (ppm): 1.54 (3H, d, J=6.6 Hz), 1.70 ∼ 2.10 (2H, m), 2.14 (2H, t, J=7.1 Hz), 4.13 ∼ 4.23 (1H, m), 4.59 (1H, q, J=6.7 Hz), 6.82 (1H, s), 7.37 (1H, s), 8.60 (1H, d, J=7.7 Hz)
       13C-NMR (75.5 MHz, DMSO-d6) δ (ppm): 21.7, 26.6, 31.2, 51.9, 54.1, 168.9, 172.8, 173.5
       MS (CI, m/e): 237 (M+ + 1)
       IR (KBr, cm-1): 1738, 1662
  • Example 2 Production of N-[2-D-(p-toluenesulfonyloxy)]propionyl-L-glutamine:
  • 47.4g (0.32 mol) of L-glutamine was added to 300 mℓ of water and 150 mℓ of toluene at room temperature and cooled to 0 to 5°C. 66 mℓ (0.32 mol) of 5N sodium hydroxide was added thereto to dissolve the L-glutamine therein. To the solution were added 90 mℓ of toluene containing 91.0g (0.32 mol) of 2-D-(p-toluenesulfonyloxy)propionyl chloride and 75 mℓ of 5N sodium hydroxide, at 0 to 5°C over a period of 2 hours, with maintaining the pH of the reaction solution to be 10. The mixture was stirred for one hour at 0 to 5°C, and toluene was removed by liquid separation. 59g of sodium chloride was added to the aqueous layer at room temperature. To the solution was added 25 mℓ of concentrated hydrochloric acid at room temperature, and the pH was adjusted to be 2.5. Then, crystal seeds were added to the solution, and the mixture was stirred for 30 minutes. Further, 8 mℓ of concentrated hydrochloric acid was added thereto so that the pH was adjusted to be 1.0. The resulting solution was allowed to stand for one hour at room temperature. The crystals formed were taken out by filtration and dried under reduced pressure to obtain 76.9g (yield: 63.8%) of N-[2-D-(p-toluenesulfonyloxy)]propionyl-L-glutamine having an optical purify of 99.6% de and melting point of 102°C.
  • The physico-chemical properties of N-[2-D-(p-toluenesulfonyloxy)]propionyl-L-glutamine were as follows:
       1H-NMR (300 MHz, DMSO-d6) δ (ppm): 1.35 (3H, d, J=6.7 Hz), 1.72 ∼ 1.98 (2H, m), 2.03 (2H, t, J=6.6 Hz), 2.43 (3H, s), 4.07 ∼ 4.14 (1H, m), 4.90 (1H, q, J=6.7 Hz), 6.83 (1H, s), 7.31 (1H, s), 7.48 (2H, d, J=8.1 Hz), 7.82 (2H, d, J=8.1 Hz), 8.44 (1H, d, J=7.8 Hz)
       13C-NMR (75.5 MHz, DMSO-d6-D2O) δ (ppm): 19.8, 22.1, 27.4, 31.9, 52.3, 76.9, 128.6, 131.1, 133.4, 146.4, 169.4, 173.6, 175.2
       MS (SIMS, m/e): 373 (M+ + 1)
       IR (KBr, cm-1): 1712, 1675
  • Example 3 Production of N-(2-D-methanesulfonyloxy)propionyl-L-glutamine:
  • 29.2g (0.20 mol) of L-glutamine was added to 185 mℓ of water and 92 mℓ of toluene at room temperature and cooled to 0 to 5°C. 40 mℓ (0.20 mol) of 5N sodium hydroxide was added thereto to dissolve the L-glutamine therein. To the solution were added 20 mℓ of toluene containing 38.0g (0.20 mol) of 2-D-methanesulfonylpropionyl chloride and 50 mℓ of 5N sodium hydroxide, at 0 to 5°C over a period of 2 hours, with maintaining the pH of the reaction solution to be 10. The mixture was stirred for one hour at 0 to 5°C, and toluene was removed by liquid separation. 76g of sodium chloride was added to the aqueous layer at room temperature. To the solution was added 21 mℓ of concentrated hydrochloric acid at room temperature, and the pH was adjusted to be 0.9. The mixture was extracted twice with 150 mℓ of chloroform/2-propanol (1/1). The organic layer was taken out by separation and concentrated to dryness to obtain 27.2g (yield: 45.8%) of N-(2-D-methanesulfonyloxy)propionyl-L-glutamine having an optical purity of 95.8% de.
  • The physico-chemical properties of N-(2-D-methanesulfonyloxy)propionyl-L-glutamine were as follows:
       1H-NMR (300 MHz, DMSO-d6) δ (ppm): 1.47 (3H, d, J=6.6 Hz), 1.77 ∼ 2.12 (2H, m), 2.15 (2H, t, J=7.5 Hz), 3.23 (3H, s), 4.17 ∼ 4.24 (1H, m), 5.07 (1H, q, J=6.6 Hz), 6.84 (1H, s), 7.35 (1H, s), 8.60 (1H, d, J=7.7 Hz)
       13C-NMR (75.5 MHz, DMSO-d6) δ (ppm): 19.4, 25.6, 31.5, 51.9, 62.6, 75.9, 169.2, 173.2, 174.4
       MS (SIMS, m/e): 297 (M+ + 1)
  • Example 4 Production of L-alanyl-L-glutamine from N-(2-D-chloro)propionyl-L-glutamine:
  • 60.0g (0.24 mol) of N-(2-D-chloro)propionyl-L-glutamine having a purity of 92.9% and 600 mℓ of 28% aqueous ammonia were put into one-liter glass autoclave for dissolution at room temperature. The resulting solution was allowed to stand at 60°C for 8 hours under the condition of an internal pressure of about 2 kg/cm2. The reaction mixture was cooled to room temperature, and concentrated under reduced pressure, and 30 mℓ of water was added to the resulting residue to make 150g as a whole. 450 mℓ of methanol was dropwise added to the resulting solution at room temperature over a period of one hour. The mixture was allowed to stand for 2 hours, and the crystals precipitated were taken out by filtration and dried under reduced pressure to obtain 35.4g (yield: 69.0%) of a crude product of L-alanyl-L-glutamine having an optical purity of 97.6% de.
  • 30g of the crude product of L-alanyl-L-glutamine was dissolved in 50 mℓ of water, 0.6g of active carbon was added thereto, and the mixture was stirred for 10 minutes at room temperature. The active carbon was removed by filtration, 42 mℓ of methanol was added to the filtrate at 30°C, and seed crystals were added thereto. Then, the mixture was allowed to stand for 2 hours. Further, 138 mℓ of methanol was added thereto at 30°C over a period of one hour and then the mixture was stirred for 2 hours. The crystals precipitated were taken out by filtration and dried under reduced pressure to obtain 26.38g (yield: 88%) of L-alanyl-L-glutamine having an optical purity of 99.9% de, a melting point (with decomposition) of 216°C, and a specific rotation [α]20 D of being -3.49° (c=10, 1N-HCl).
  • Example 5 Production of L-alanyl-L-glutamine from N-(2-D-bromo)propionyl-L-glutamine:
  • In 300 mℓ of 28% aqueous ammonia, was dissolved 20.0g (0.07 mol) of N-(2-D-bromo)propionyl-L-glutamine at room temperature and the solution was allowed to stand for 20 hours at room temperature. The reaction mixture was concentrated under reduced pressure, and about 6 mℓ of water was added to the resulting residue to make 40g as a whole. 126 mℓ of methanol was dropwise added to the resulting solution at room temperature over a period of one hour, and the mixture was allowed to stand for 2 hours. The crystals thus formed were taken out by filtration and dried under reduced pressure to obtain 12.1g (yield: 78.1%) Of a crude product of L-alanyl-L-glutamine having an optical purity of 98.9% de.
  • 11.0g of the crude product of L-alanyl-L-glutamine was dissolved in 18.3 mℓ of water, 0.22g of active carbon was added thereto, and the mixture was stirred for 10 minutes at room temperature. The active carbon was removed by filtration, 15.4 mℓ of methanol was added to the resulting filtrate at 30°C, and seed crystals were added thereto. Then, the mixture was allowed to stand for 2 hours. Further, 50.6 mℓ of methanol was added thereto at 30°C over a period of one hour, and the mixture was stirred for 2 hours. The crystals precipitated were taken out by filtration and dried under reduced pressure to obtain 9.84g (yield: 89.5%) of L-alanyl-L-glutamine having an optical purity of 99.8% de.
  • Example 6 Production of L-alanyl-L-glutamine from N-[2-D-(p-toluenesulfonyloxy)]propionyl-L-glutamine:
  • In 300 mℓ of 28% aqueous ammonia, was dissolved 30.0g (0.08 mol) of N-[2-D-(p-toluenesulfonyloxy)]propionyl-L-glutamine at room temperature, and the solution was allowed to stand for 24 hours at room temperature. The reaction mixture was concentrated under reduced pressure, and about one ml of water was added to the resulting residue to make 50g as a whole. 200 mℓ of methanol was dropwise added to the resulting solution at room temperature over a period of one hour, and the mixture was allowed to stand for 2 hours. The crystals thus formed were taken out by filtration and dried under reduced pressure to obtain 8.6g (yield: 49.1%) of a crude product of L-alanyl-L-glutamine having an optical purity of 99.3% de.
  • 8g of the crude product of L-alanyl-L-glutamine was dissolved in 13.3 mℓ of water, 0.16g of active carbon was added thereto, and the mixture was stirred for 10 minutes at room temperature. The active carbon was removed by filtration, 11.2 mℓ of methanol was added to the resulting filtrate at 30°C, and seed crystals were added thereto. Then, the mixture was allowed to stand for 2 hours. Further, 36.8 mℓ of methanol was added thereto at 30°C over a period of one hour and then the mixture was stirred for 2 hours. The crystals precipitated were taken out by filtration and dried under reduced pressure to obtain 7.41g (yield: 92.6%) of L-alanyl-L-glutamine having an optical purity of 99.9% de.
  • Example 7 Production of L-alanyl-L-glutamine from N-(2-D-methanesulfonyloxy)propionyl-L-glutamine:
  • In 156 mℓ of 28% aqueous ammonia, was dissolved 15.6g (0.053 mol) of N-(2-D-methanesulfonyloxy)propionyl-L-glutamine at room temperature, and the solution was allowed to stand for 46 hours. The reaction mixture was concentrated under reduced pressure, and about 7 mℓ of water was added to the resulting residue to make 35g as a whole. 100 mℓ of methanol was dropwise added at room temperature over a period of one hour and the mixture was allowed to stand for 2 hours. The crystals thus formed were taken out by filtration and dried under reduced pressure to obtain 5.78g (yield: 50.5%) of a crude product of L-alanyl-L-glutamine having an optical purity of 96.4% de.
  • 5.0g of the crude product of L-alanyl-L-glutamine was dissolved in 8.3 mℓ of water, 0.1g of active carbon was added thereto, and the mixture was stirred for 10 minutes at room temperature. The active carbon was removed by filtration, 7.0 mℓ of methanol was added to the resulting filtrate at 30°C, and seed crystals were added thereto. Then, the mixture was allowed to stand for 2 hours. Further, 23.0 mℓ of methanol was added thereto at 30°C over a period of one hour and then the mixture was stirred for 2 hours. The crystals precipitated were taken out by filtration and dried under reduced pressure to obtain 4.30g (yield: 86.0%) of L-alanyl-L-glutamine having an optical purity of 99.0% de.
  • Example 8 Amination of N-(2-D-bromo)propionyl-L-glutamine:
  • In 3 mℓ of 28% aqueous ammonia, was dissolved 300 mg (1.07 mmol) of N-(2-D-bromo)propionyl-L-glutamine having an optical purity of 97.9% de at room temperature, and the solution was allowed to stand for 20 hours at room temperature, in the same manner as in Example 5. The reaction mixture was concentrated under reduced pressure and ammonia was removed therefrom by evaporation. By HPLC analysis under the following condition, formation of 202 mg (yield: 87.1%) of L-alanyl-L-glutamine having an optical purity of 98.2% de was confirmed.
    Condition for HPLC Analysis:
  • Column: YMC-pack, ODS-AQ313
  • Mobile Phase: 0.01M KH2PO4
  • Detection: UV 210 nm
  • Separately, N-(2-D-bromo)propionyl-L-glutamine was aminated according to the method described in Hoppe-Seyler's Z. Physiol. Chem., 105, 58 (1919):
  • In 2 mℓ of 26.7% aqueous ammonia, was dissolved 300 mg (1.07 mmol) of N-(2-D-bromo)propionyl-L-glutamine having an optical purity of 97.9% de at room temperature. The solution was allowed to stand for one hour in water bath of 100°C. The reaction mixture was concentrated under reduced pressure and ammonia was removed therefrom by evaporation. By HPLC analysis under the same condition as mentioned above, formation of 147 mg (yield: 63.4%) of L-alanyl-L-glutamine having an optical purity of 96.8% de was confirmed.
  • Example 9 Production of N-(2-D-bromo)propionyl-L-glutamine:
  • 21.9g (0.15 mol) of L-glutamine was added to 300 mℓ of water and 75 mℓ of toluene at room temperature, and the mixture was cooled to 0 to 5°C. 30 mℓ (0.15 mol) of 5N sodium hydroxide was added thereto to dissolve L-glutamine therein. 30 mℓ of toluene containing 25.7g (0.15 mol) of 2-D-bromopropionyl chloride was added to the resulting solution at 0 to 5°C over a period of 2 hours, while maintaining the pH of the reaction solution to be 10 by dropwise adding 25 mℓ of 5N sodium hydroxide thereto. The mixture was stirred for one hour at 0 to 5°C, toluene was removed by liquid separation, and 40g of sodium chloride was added to the aqueous layer at room temperature.
  • 15 mℓ of concentrated hydrochloric acid was added to the resulting solution at room temperature so as to adjust the pH to be 1.0, and the solution was allowed to stand for one hour at room temperature. The crystals formed were taken out by filtration and dried under reduced pressure to obtain 40.4g (yield: 95.8%) of N-(2-D-bromo)propionyl-L-glutamine having an optical purity of 97.9% de and a melting point of 142°C.

Claims (2)

  1. A process for producing alanylglutamine, which comprises reacting an N-(2-substituted)-propionylglutamine compound represented by the formula (I):
    Figure 00210001
    where X represents halogen, alkylsulfonyloxy, or substituted or unsubstituted arylsulfonyloxy, with ammonia at a temperature of 60°C or below.
  2. N-(2-substituted)propionylglutamine compound represented by the formula (I'):
    Figure 00210002
    where X1 represents chlorine, or a salt thereof.
EP19930117578 1992-10-29 1993-10-29 Process for producing alanylglutamine Expired - Lifetime EP0595345B1 (en)

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AU3927400A (en) * 1999-04-01 2000-10-23 University Of Virginia A process for the production of glutamine derivatives and glutamine containing molecules
US6649746B1 (en) 1999-05-07 2003-11-18 University Of Virginia Patent Foundation Biological production of stable glutamine, poly-glutamine derivatives in transgenic organisms and their use for therapeutic purposes
SE0201713D0 (en) 2001-11-23 2002-06-06 Gramineer Internat Ab New methods and use III
DE10161077A1 (en) * 2001-12-12 2003-06-18 Boehringer Ingelheim Vetmed Highly concentrated stable meloxicam solutions for needleless injection
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CN101062938B (en) * 2006-04-25 2010-09-01 福建三爱药业有限公司 Preparation method of N(2)-L-alanyl-L-glutamine
US9101529B2 (en) * 2009-10-12 2015-08-11 Boehringer Ingelheim Vetmedica Gmbh Containers for compositions comprising meloxicam
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CN102093250B (en) * 2010-12-02 2013-03-20 海南本创医药科技有限公司 Refining method of alanyl-glutamine compound
CN102260189B (en) * 2011-05-31 2014-05-21 四川科伦药物研究有限公司 Method for preparing key intermediate N(2)-D-2-propionyl chloride-L-glutamine of N(2)-L-alanyl-L-glutamine
CN103012550B (en) * 2011-09-26 2014-12-10 重庆莱美药业股份有限公司 Preparation method of N (2)-L-alanyl-L-glutamine
CN103012191B (en) * 2011-09-26 2015-04-22 重庆莱美药业股份有限公司 Method for preparing D-2-substituted propionyl-L-glutamine
CN102731613B (en) * 2012-07-18 2013-12-11 济南诚汇双达化工有限公司 Preparation method of L-alanyl-L-glutamine
CN103910780A (en) * 2014-04-09 2014-07-09 上海皓骏医药科技有限公司 Preparation method of L-alanine-L-glutamine compound
CN106701869A (en) * 2016-12-30 2017-05-24 山东辰龙药业有限公司 Preparation method of N (2)-L-alanyl-L-glutamine

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CN102491918A (en) * 2011-11-28 2012-06-13 海南灵康制药有限公司 Alanyl glutamine compound and preparation method thereof
CN102491918B (en) * 2011-11-28 2013-07-10 海南灵康制药有限公司 Alanyl glutamine compound and preparation method thereof
US20150038740A1 (en) * 2011-11-28 2015-02-05 Hainan Lingkang Pharmaceutical Co., Ltd. Alanyl glutamine compound and preparation method thereof
US9024064B2 (en) * 2011-11-28 2015-05-05 Hainan Lingkang Pharmaceutical Co., Ltd. Alanyl glutamine compound and preparation method thereof

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ES2141748T3 (en) 2000-04-01
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US5380934A (en) 1995-01-10
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